Stability of platelet surface antigens during storage

We measured changes in A, B, 2H, PlA1, and HLA Class I antigens on human platelets stored as routine platelet concentrates (PCs) in 50 to 60 ml of citrate-phosphate-dextrose-adenine (CPDA-1) plasma in polyolefin (PL 732) bags at 22 degrees C with continuous cartwheel rotation. Samples were obtained at 1, 3, 5, and 10 days of storage; incubated with human IgG anti-A, -B, -HLA and -PlA1; incubated with mouse monoclonal 125I-labeled anti-human IgG; centrifuged through phthalate ester oils; and assayed in a gamma scintillation counter. Additionally, group O platelets were analyzed using 125I-labeled IgM mouse monoclonal anti-Type 2H. Mean values for molecules of Ig bound per platelet showed that platelet surface antigens A, B, 2H, PlA1, and HLA Class I showed no significant change during 10-day storage as routine PCs in CPD-A1 in PL 732 bags. Identical radioassays were performed with platelets incubated at 22 degrees C in plastic test tubes for 24 hours in homologous plasma from donors negative for the respective antigens and in a variety of artificial media with albumin and lipids. No significant changes occurred in any of the surface antigens, except for the loss of approximately 50 percent of the blood group A antigen from platelets stored in O plasma or in albumin media. These data indicate that HLA, PlA1, and type 2H structures do not readily dissociate from the platelet membrane during storage, while some blood group A antigens, presumably acquired passively from the plasma, will elute from the platelet under certain conditions. Routine storage conditions are unlikely to alter the immunogenicity of platelets due to a loss of antigen expression.     

The detection of platelet alloantibodies by flow cytometry

A flow cytometric procedure was investigated for its ability to detect antibodies directed against blood group A, HLA, and PlA1 (HPA-1a) antigens. When type O sera were tested against platelets from blood group A donors, only 9 of 14 positive reactions were observed. Furthermore, the expression of blood group A varied more than 100-fold on platelets derived from individual donors. When anti-HLA-A2 and -B7 were evaluated, 11 of 11 individuals with HLA-A2 and -B7 antigens reacted. In contrast, when platelets from donors whose HLA antigens included HLA-B8 or -B12 were tested with anti-HLA-B8 or -HLA-B12, respectively, positive reactions were observed in only 3 of 7 instances, despite the fact that the lymphocytes reacted strongly. Platelets from 10 HLA-A2-positive donors, which had been stored for up to 20 months at -70 degrees C, were studied. In all cases, frozen-stored platelets reacted well with an anti-HLA-A2. Limited testing with an anti-PlA1 (anti-HPA-1a) showed equal reactivity with fresh and frozen platelets. Finally, the method was compared to a visual immunofluorescence assay using sera from patients who were refractory to platelet transfusions. The results agreed in 30 of 37 comparisons, and most discrepancies were resolved in favor of flow cytometry. It is concluded that flow cytometry is useful for detecting platelet alloantibodies and possibly for prospective platelet crossmatching, as HLA- and platelet-specific antibodies can be identified by using platelets stored frozen for several months.     

Flow cytometric analysis in platelet crossmatching using a platelet suspension immunofluorescence test

BACKGROUND: The sensitivity of flow cytometric measurement of platelet antibodies in a crossmatch technique was investigated. STUDY DESIGN AND METHODS: The corrected count increment after platelet transfusion was compared with the fluorescence ratio determined by flow cytometric measurement. RESULTS: When crossmatching was performed before transfusion(s) in alloimmunized patients, a fluorescence ratio < or = 1.7 was associated with satisfactory responses (corrected count increment > or = 7.5), and the predictive values for negative and positive crossmatch results were 94 and 87 percent, respectively. Analysis of antigen preservation during platelet storage with antibodies to HLA alpha-chain, HLA-B27, HPA-1a, and HPA-3a showed that platelets can be stored, refrigerated, for up to 4 weeks without significant loss of HLA class I and HPA-1a. There was a slight but continuous loss of HPA-3a upon storage. CONCLUSION: Flow cytometric measurement of fluorescence in the platelet suspension immunofluorescence test can be used for platelet crossmatching, with sensitivity and predictive values comparable to those of previously described techniques and with the advantage of automation.     

Independent roles for platelet crossmatching and HLA in the selection of platelets for alloimmunized patients

BACKGROUND: Although HLA-matched platelets are frequently requested for alloimmunized patients, recent evidence has indicated that 1-hour posttransfusion platelet increments in these patients are specifically sensitive to crossmatch compatibility. STUDY DESIGN AND METHODS: To determine the extent of advantage gained by use of single-donor apheresis (SD) platelets selected on the basis of HLA match when crossmatch-compatible SD platelets were available, a total of 220 platelet transfusions given in the absence of individually determined significant nonimmune factors were analyzed in a well-characterized cohort of platelet-refractory patients. Platelets were selected by solid-phase crossmatch from a small donor pool of relatively poor HLA matches or, upon request, ordered as HLA-matched and later crossmatched. RESULTS: Alloimmunized patients responded better to SD platelets selected on the basis of HLA than to pooled platelet concentrates or SD platelets selected at random, although most of the benefit was limited to the 57-percent subset of good HLA matches. Crossmatch-compatible SD platelets provided similar posttransfusion platelet increments independent of the HLA match. None of 31 crossmatch- incompatible SD platelets transfused provided an adequate increment, including 13 that were ordered as HLA-matched platelets. CONCLUSION: No benefit could be demonstrated from requesting that SD platelets be HLA- matched when crossmatch-compatible SD platelets were available.     

The predictive value of crossmatching platelet transfusion for alloimmunized patients

To determine the predictive value of a radiolabeled antiglobulin test for platelet crossmatching to select platelets for transfusion, we evaluated 230 transfusions in 42 alloimmunized oncology patients. Transfusions were classified according to the degree of HLA match. The value of the crossmatch in predicting platelet transfusion outcome was analyzed for each HLA category. For predicting the 1-hour corrected platelet count increment of all transfusions, the sensitivity of the test was 86 percent, specificity 84 percent, and total predictive value 84 percent. The crossmatch technique had similar predictive ability in all categories of HLA match, whether analyzing the results on the basis of the 1- or 24-hour platelet count rise.     

青岛地区已知HLA和HPA分型血小板供者库的建立及临床应用

目的建立青岛市已知HLA和HPA分型的无偿机采血小板供者库并用于解决血小板输注无效(PRT)患者的血小板输注。方法采用PCR-SSO流式磁珠法和PCR-SSP法分别对青岛地区无偿机采血小板捐献者1 800人进行HLA-Ⅰ类(A,B位点)、HPA-1—-5,-15基因分型,针对不同免疫状态的PTR患者比较3种不同配型法(血小板随机交叉配型法、抗原特异性配合法、抗原阴性选择法)解决血小板输注无效的效果。结果在高致敏(群体反应性抗体PRA>60%)患者中,HLA和或HPA特异性配型效果较好;轻度致敏(PRA<60%)患者随机交叉,抗原阴性选择,抗原特异性配型方案效果相似。不考虑ABO血型的因素1 800名的血小板供者库可以使99%的患者在库中找到至少1例HLA完全相合的供者,理论估算的供者库规模被不同表型的患者库中检索结果证实。结论理论上青岛本地化已知分型血小板供者库规模需达到6 000人左右可以有效解决血小板输注无效患者的血小板输注问题。只有重度致敏的患者需要HLA完全相同的供者,而轻度致敏的患者可以选择随机交叉配合或抗原阴性选择方案。     

Racial differences in the availability of human leukocyte antigen—Matched platelets

In view of the demonstrated difficulty of providing African American patients with compatible red cells and bone marrow from the predominantly white donor population in the United States, it should be determined if African American patients have greater difficulty obtaining human leukocyte antigen (HLA)-matched platelets. Twenty-two alloimmunized African American patients and 20 alloimmunized white patients were studied. These groups were HLA-matched against our hospital pheresis service (1,157 donors), the local Red Cross pool (3,405 donors), and the combined population (4,562 donors). The frequency of each patient's HLA phenotype was calculated in the white population and in the African American population. More African Americans did not have a well matched (A or BU) platelet donor. Fewer African American patients had sufficient matched (A and B) donors to support the average period of aplasia. On average, the African American patients had half as many matched donors as compared to the White patients. Sixty-eight percent of African American patients had HLA types which were more difficult to find in a white population. Ninety percent of white patients had HLA phenotypes which were more common in the white population. We have shown that it is more difficult to obtain HLA-matched platelets for alloimmunized African American patients as compared to alloimmunized white patients. With larger donor pools, platelet support for African American patients is easier, but uncommon HLA types remain. Our results support efforts to enlarge the donor pool with an emphasis on specific recruitment programs aimed at the African American community. © 1996 Wiley-Liss, Inc.     

Selecting donors of platelets for refractory patients on the basis of HLA antibody specificity

BACKGROUND: Patients who are refractory to platelet transfusion as a result of HLA alloimmunization are generally given HLA-matched or crossmatched platelets. However, HLA-matched platelets that are matched at HLA-A and -B loci (A-matched) or those without any mismatched or cross-reactive antigens (BU-matched) are frequently unavailable. A disadvantage of crossmatching is that crossmatched platelets have a shelf life of only 5 days, so that crossmatch tests must be performed frequently for patients requiring long-term platelet transfusions. An alternative method is the selection of platelets according to the patient's HLA antibody specificity, called the antibody specificity prediction (ASP) method. STUDY DESIGN AND METHODS: An anti-human globulin-enhanced microlymphocytotoxicity test modified by a double addition of serum and a computer program were used to determine the specificity of patients' HLA antibodies. Platelet crossmatching was performed with a solid-phase adherence assay. The percentage of platelet recovery (PPR) was determined in 1621 platelet transfusions in an observational study in 114 patients, and the PPR of platelets selected by the ASP method was compared with the PPR of those that were HLA-matched, crossmatched, or randomly selected. The numbers of potential donors in files of HLA-typed donors as identified by HLA matching vs. the ASP method were determined. RESULTS: After adjustments for covariates, the mean +/- SEM PPR was similar for HLA-matched (21 +/-4%), cross-matched (23+/-4%), and ASP-selected (24+/-3%) platelets and was significantly lower for randomly selected (15+/-1.4%) platelets. For 29 alloimmunized HLA-typed patients, the mean number of potential donors found in a file of 7247 HLA-typed donors was 6 who were an HLA-A match (median = 1), 33 who were an HLA-BU match (median = 20), and 1426 who were identified by the ASP method (median = 1365). CONCLUSION: The ASP method of donor selection for refractory alloimmunized patients appears as effective as HLA matching or crossmatching. Far more donors are identified in a file of HLA-typed donors by the ASP method than by HLA matching, and this indicates that the ASP method provides important advantages regarding the availability of compatible platelet components.     

The relationship between donor-recipient lymphocytotoxicity and the transfusion response using HLA-matched platelet concentrates

Matching donor-recipient pairs for HLA antigens provides a logical starting point for selecting platelet donors for thrombocytopenic recipients who have demonstrated refractoriness to pooled random donor platelet transfusions. However, not all recipients achieve a compatible transfusion response with platelets selected by HLA type, indicating that additional selection methods are required. Because of studies indicating that a positive lymphocyte cross-match assay between recipient serum (antibody) and donor lymphocytes predict acute renal allograft rejection, it was hoped that this assay might be useful in predicting the platelet transfusion response for alloimmunized patients. The data herein indicate that the results of this assay in no way correlate with the platelet transfusion response. The lymphocyte cross-match would seem to be helpful in the selection of donors where known HLA antigen mismatches occur between donor and recipient. However, even in this situation, no correlation was observed between the transfusion response and donor-recipient lymphocytotoxicity reaction. Clearly additional assays are needed for the selection of compatible donors from those matched by HLA antigens.     

Generation of annexin V-positive platelets and shedding of microparticles with stimulus-dependent procoagulant activity during storage of platelets at 4 degrees C

BACKGROUND: The ability of propyl gallate to activate platelet factor 3 has been determined through the activated partial thromboplastin time, but its effect on phosphatidylserine has not been established. STUDY DESIGN AND METHODS: A novel platelet activator, propyl gallate, was introduced to a study of platelets stored at 4 degrees C. The effects of storage on platelet coagulation activity, on phosphatidylserine, and on the shedding of activated and activable membrane particles (microparticles) were examined by activated plasma clotting time, and the effect on annexin V binding was examined by gated flow cytometry. The ratios of annexin V binding and microparticle shedding in stored platelet samples were compared with those in fresh platelets stimulated with propyl gallate. RESULTS: Microparticle shedding by stored platelets compensated for the diminished procoagulant potential of intact platelets (shown as the total propyl gallate-dependent platelet factor 3 activity), which did not change during prolonged (20-day) storage, but levels of phosphatidylserine confined to microparticles increased dramatically as platelet counts fell. Both annexin V binding and microparticle shedding increased spontaneously with storage and artificially with propyl gallate stimulation. However, at the same level of annexin V binding, stored platelets shed more microparticles than did fresh platelets stimulated with propyl gallate. CONCLUSION: Propyl gallate induces platelet procoagulant activity and annexin V binding. Stored platelets differ from fresh platelets in a lower reactivity to propyl gallate activation and a higher rate of microparticle shedding.     

HLA antibody enhancement by double addition of serum: use in platelet donor selection

The identification of HLA class I alloantibodies is important for organ transplantation and platelet transfusion in alloimmunized patients. Because microcytotoxicity testing against frozen trays of lymphocyte panels is rapid and efficient for determining specificities of unknown antibodies, a simple method was devised to increase test sensitivity to weak antibodies. Standard anti-human globulin (AHG)-facilitated microcytotoxicity was modified by the insertion of a double addition-of-serum (DAS) step, and reagent and patient's sera were evaluated by both methods. DAS modification increased antibody titers and, more significantly, made the identification of weak specificities easier because of the twofold to threefold increase in reactivity rates (29-42% for AHG vs. 75-82% for DAS) of panel cells that were expected to be positive, while low (approx. 1%) "extra" reaction rates were maintained for cells that were expected to be negative. DAS was relatively unaffected by variations in serum volumes or target cell preparation, and its use did not significantly increase test time or costs. In a program of platelet donor selection driven by donor antibody rather than donor-recipient antigen matching, DAS greatly facilitated platelet transfusion support for alloimmunized patients.     

Extension of platelet concentrate storage by addition of sodium bicarbonate

Viability of platelet concentrate (PC) stored in polyvinylchloride bags in an elliptical rotator at 22 degrees C (standard PC) was assessed by in vitro tests, and an alternate approach to extending the shelf-life of PC by the addition of hypertonic sodium bicarbonate (test PC) was investigated. The fall in the pH which occurred during storage in standard PC was arrested in test PC. Furthermore, platelets stored under these test conditions maintained their morphology better than in standard PC as judged by their mean platelet volume and platelet distribution width. Recovery of stored platelets from hypotonic shock at 37 degrees C following resuspension in fresh plasma was better for test platelets. Results indicated that platelets in standard PC were viable up to day 3 but were not viable at day 7. Platelets store better in PC to which sodium bicarbonate has been added and behave as viable platelets up to 7 days.     

Detection of cytomegalovirus antibody in stored blood products using latex agglutination

The detection of antibody to cytomegalovirus (CMV) in donor sera is one effective method for the prevention of posttransfusion CMV infection in seronegative recipients. A passive latex agglutination test (CMV Scan, Hynson, Wescott & Dunning, Baltimore, MD) has been shown to be an acceptable method of screening sera from the blood donor population. The procedure for this test, however, does not permit the testing of stored donor blood products. To evaluate the feasibility of testing blood components at various times during storage, the authors examined 25 CMV-positive and 25-CMV negative samples of CPDA-1 plasma from 50 units each of platelets and red cells. Plasma samples from platelets stored at 22 degrees C were tested on each of the 5 days of storage. Samples from red cell units stored at 2 to 6 degrees C were tested on storage Days 1, 2, 4, 6, 8, 10, 12, and 14. Of 250 tests done on plasma from platelet units, there were 123 true-positive and 123 true-negative results (sensitivity and specificity, 98.4%). Of 400 tests on plasma from red cell units, there were 200 true-positive and 198 true-negative results (sensitivity, 100%; specificity, 99.5%). These data show that the CMV Scan test can be used reliably to test segments of CPDA-1 plasma from platelets stored for up to 5 days and from red cells stored for up to 14 days.     

Potential HLA-matched platelet donor availability for alloimmunized patients

An analysis of the computer searches for HLA-matched donors was done for 100 alloimmunized patients requiring HLA-matched platelet transfusion support. With a pool of 2470 donors, an average of only 1.3 (range 0–14) perfectly matched donors were potentially available per patient. Patients had an average of 9 donors with no mismatched antigens. Although the number of potential donors increased when cross reactive or single antigen mismatches were considered, our estimate of donor availability of this type is lower than reported previously. More donors were identified for the 39 patients with relatively common genotypes than for the remaining 61 patients. Of the latter group, 25 patients did not have any potential donors with "no mismatches" while in most of the others the only suitable donor was a family member. These data suggest that, given the limitations of platelet donor selection using current HLA typing technology, it may be difficult to provide long-term platelet transfusion support for alloimmunized patients with unusual HLA types. This fact should be considered when planning intensive therapeutic regimens for alloimmunized patients with leukemia.     

Stability of platelet and plasma HLA concentrations in healthy adults or random-donor platelet concentrates

The quantitative differences between the platelet HLA antigens of different individuals are potentially significant in determining the survival of transfused platelets in allosensitized patients. To learn whether platelet HLA concentration is sufficiently stable to be documented for each donor and used for donor selection, platelet and plasma HLA concentrations in five healthy adults were measured monthly for 5 consecutive months. HLA concentrations were measured by using a competitive enzyme-linked immunoassay. The results showed that both platelet and plasma HLA concentrations varied within +/- 20 percent of the mean values in each individual. Changes in plasma and platelet HLA concentrations were also studied in 5 units of random-donor platelets during in vitro storage. HLA concentrations were determined every other day over a 5-day period. There was no significant quantitative change in the platelet and plasma HLA concentrations during the 5 days of in vitro storage. Thus, platelet HLA concentrations in healthy individuals are stable and may be quantitatively documented for donor selection.     

Seven-day storage of apheresis platelets: report of an in vitro study

BACKGROUND: The objective of this study was to determine the allowable platelet content limits for apheresis platelets stored for 7 days in a platelet storage bag (COBE ELP, Gambro BCT). METHODS: Apheresis platelets under controlled concentration and volume per bag were stored in plasma up to 8 days at 22 degrees C with horizontal agitation. Routinely evaluated in vitro platelet parameters were followed. Oxygen consumption was directly measured with a Clark-type electrode. All components were cultured in aerobic medium on Day 7. RESULTS: Twenty-four components were evaluated in storage configurations (median [range], 340 [110-402] mL, 1.32 [0.99-2.45] x 10(6) platelets/microL, and 4.8 [1.4-5.9] x 10(11) platelets/bag). No bacterial contamination was detected. One component had a pH value at 22 degrees C of below 6.0 before Day 5 with attendant loss of all other in vitro function measures. The pH value at 22 degrees C was maintained above 6.2 for the remaining 23 components. A pH value of greater than 7.4 was observed at some point in storage for 13 of 23 units, although platelet function or activation was not adversely affected. Aerobic metabolic function was maintained over 7 days with O2 consumption of 321 micromol per hour per 10(12) platelets on Day 7. CONCLUSION: Although a continuing decline of platelet in vitro characteristics can be observed for storage beyond 5 days, apheresis platelets in plasma stored 100 to 400 mL per bag, 1.0 x 10(6) to 2.5 x 10(6) platelets per microL, and a maximum of 5.1 x 10(11) platelets per bag maintained in vitro platelet characteristics over 7 days of storage.     

A cost-effectiveness evaluation of platelet crossmatching and HLA matching in the management of alloimmunized thrombocytopenic patients

Effective platelet support for alloimmunized refractory thrombocytopenic patients may be provided by several potential strategies, the most common being HLA-matched single-donor platelets or crossmatch-compatible, pooled random- or single-donor platelets. This study used a detailed economic analysis to compare the cost-effectiveness of several techniques for platelet crossmatching and that of HLA-matched single-donor platelets. The crossmatch methods evaluated were a microlymphocytotoxicity test (LCT), an immunofluorescence technique (PSIFT), a radioactive antiglobulin test (PRAT), and an enzyme-linked immunosorbent assay (ELISA). The analysis was based on the need to support 100 refractory patients with acute leukemia with a presumed requirement of 500 transfusions. The relative costs for a successful crossmatch were: PRAT less than LCT less than LCT + PRAT less than PSIFT less than ELISA. In the comparison of the crossmatch methods, an increase in costs was generally associated with an increase in the number of successful transfusion episodes. However, decreasing marginal gains were seen. The HLA-matched single-donor platelets were relatively cost-inefficient in comparison to the crossmatch-compatible platelets. A theoretic sequence of tests for cost-effective provision of optimal platelet support in refractory patients was evaluated. Such considerations of cost are important in the selection of an optimal program for the management of alloimmunized refractory thrombocytopenic patients.     

Cryopreservation of single-donor platelets with a reduced dimethyl sulfoxide concentration by the addition of second-messenger effectors: enhanced retention of in vitro functional activity

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. This creates an inventory problem, which could be overcome by the use of cryopreservation to allow long-term storage of platelets. It has been demonstrated that the addition to platelets of a mixture of second- messenger effectors (platelet storage solution), allows these cells to retain significant in vitro functional activity following cold storage. Analysis is needed of the ability of this second messenger effector mixture both to protect platelets during cryopreservation and to reduce the need for a cryoprotectant. STUDY DESIGN AND METHODS: Fresh single- donor platelet units (n = 8) were divided into three samples and treated with 6-percent dimethyl sulfoxide (DMSO), 2-percent DMSO or the platelet storage solution and 2-percent DMSO. The samples were placed directly into a -80 degrees C freezer and stored for 1 week, after which they were thawed and analyzed for in vitro functional activity. RESULTS: Platelets cryopreserved with the platelet storage solution and 2-percent DMSO displayed statistically higher retention of functional activity and viability–including cell number, percent of discoid cells, extent of shape change, and hypotonic shock response–than did platelets stored by the method using 6-percent DMSO. In addition, the treated platelets displayed statistically lower expression of p- selectin. The treated platelets showed no loss of cell number, > 88- percent retention of discoid morphology, and > 75-percent retention of ristocetin-induced aggregation as compared to values for these measures in fresh platelets. CONCLUSION: The use of this platelet storage solution in the cryopreservation of platelets yields a significant improvement in their postthaw in vitro recovery and allows for a reduction of the DMSO concentration from 6 to 2 percent, with superior maintenance of in vitro viability and function.     

Correlation of in vivo and in vitro functions of fresh and stored human platelets

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.     

Effects of storage conditions on platelet cytoskeletal proteins

The platelet cytoskeleton is a major determinant of platelet morphology and function. Changes in the protein composition of the cytoskeleton were studied during storage of platelets at 20 degrees to 24 degrees C under blood banking conditions. Cytoskeletons were prepared by extraction of washed platelets with the detergent Triton X-100 and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the three major proteins visible with Coomassie blue staining--actin, myosin, and actin-binding protein--silver staining revealed other proteins associated with the cytoskeletons of freshly collected and stored platelets. The resting platelet cytoskeleton contained 8% to 10% of the total platelet protein and approximately 50% of the total actin. During storage of platelet concentrates for up to five days in the PL 732 container, proteins of 50,000 to 55,000 and 90,000 mol wt were increasingly incorporated into the Triton-insoluble fraction, whereas the amounts of cytoskeleton-associated actin-binding protein, myosin, and actin were maintained at levels present in fresh platelets. Storage of platelets under conditions that allowed the reduction of platelet concentration pH to nearly 6.0 resulted in a marked decrease in the amounts of the major proteins of the cytoskeleton. The loss of specific proteins from platelets stored for extended periods with reduced pH, accompanied by the appearance of lower molecular weight proteins in the cytoskeleton, suggests that extensive proteolysis may occur under certain storage conditions. These data show that the conditions employed for storage of platelet concentrates influence the protein composition of the cytoskeleton and the total protein content of the platelet.